I was trying to extract metabolites from streptomyces by using normal phase column chromatography with EtOAc : hexane (gradient) as solvent system. After sometime I could see a coloured substance moving down from the column. I collected some of it, but most of it is stuck to the silica and not eluting. I tried using solvents with higher polarity than EtOAc. Also used water but all of it is not coming off the silica. Water was able to remove some of it but not all.. Does it have to do anything with the acidic nature of silica gel? Suggestions please!!!
Try mixture of MDC and methanol or Chloroform and Methanol or Chloroform, Methanol and Water
You tried what I would suggest- used solvents with a higher polarity, including water. I am assuming you already tried dichloromethane and methanol already. What is left on the column is very polar and might be bound to the silica. Consider running acidic water. If the compound is basic, it will interact with the acid in the water. The acid will also make the solution even more polar too (ions from acid). I like to use formic or acetic acid.
I'd consider running a basic polar solution as a last resort, since silica is soluble in water (or methanol) at high pH (starts dissolving at pH 7.5 or 8, depending on which reference you read). You would then need to recover your compound from the solution of dissolved silica. Ammonium hydroxide is what I'd use for this situation, and do not run this solution through an automated flash system. The silica may precipitate and clog the lines after the column. Again, I would try polar basic only as a last resort.
i recommend you to try in TLC prior to column chromatography.
Are you extracting from culture medium or from cells?
From culture broth is better to use octadecilsilica (silica C-18, reversed phase) to concentrate and purify more polar compounds.
If you are extracting cell pellet with organic solvent (dichloromethane, ethyl acetate, etc.) you can use silica (normal phase). If your compounds do not elute try with recommendations given. Best regards
I agree with Sebastians suggestions . I think you can try the TLC Work up of the sample. But try to purify your extract further by some more solvent extractions . More impure / crude extract you have , more problems you get into during any even open column chromatography (CC) . so you my dear can proceed as follows ,
Purify this extract further by partition solvent extractions , you may get may be more than 2 extracts. Check each of this on TLC to see its purity / for no of main metabolites in them . It will be very helpful if you already know the metabolites, & have their standards with you , then try to run the standard on the TLC & compare the amounts of your metabolites in each extract . Try to separate well these metabolites on the TLC, then run a 15-25 % more concentrated solvent system for the CC . I assure you will get the better results . Then keep collecting the fractions , separate them through TLC identifications runs, then merge the fractions with same Rf Values , evaporate / concentrate them for further characterization by the other Chemical / instrumental techniques. Dr Abid Aziz Ajaz.
Sounds like you may have phenolics or acids in your extract. When I would encounter this I would either acidified the solvents I was using for flash chromatography, e.g. 1-5% acetic or pref. TFA as it has a lower bp. Obviously doing this is a risk if you have an acid sensitive compound. Alternatively make prep. TLC plates with 2% oxalic acid. Following elution you need to wash your organic extracts with water to ensure removal of any traces of oxalic acid. This works well for quinones, phenolics and some acids.
Aisha Tabassum
I'll answer this here:
Plant extract stucked in silica column ( column chromatography) ? | ResearchGate
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